ABSTRACT According to general belief, the conformational information on short linear peptides in solution derived at ambient temperature from NMR spectrometry represents a population-weighted average over all members of an ensemble of rapidly interconverting conformations. Usually the search for discrete conformations is concentrated at low temperatures especially when sharp NMR resonances are detected at room temperature. Using the peptide Ac-RGD-NH(2) (Ac-Arg-Gly-Asp-NH(2), Ac: acetyl) as a model system and following a new approach, we have been able to demonstrate that short linear peptides can adopt discrete conformational states in DMSO-d(6) (DMSO: dimethylsulfoxide) which vary in a way critically dependent on the reconstitution conditions used before their dissolution in DMSO-d(6). The conformers are stabilized by intramolecular hydrogen bonds, which persist at high temperatures and undergo a very slow exchange with their extended structures in the NMR chemical shift time scale. The reported findings provide clear evidence for the occurrence of solvent-induced conformational exchange and point to DMSO as a valuable medium for folding studies of short linear peptides.

[Show abstract][Hide abstract]ABSTRACT: The ability of an integrin to distinguish between the RGD-containing extracellular matrix proteins is thought to be due partially to the variety of RGD conformations. Three criteria have been proposed for the evaluation of the structure-activity relationship of RGD-containing peptides. These include: (i) the distance between the charged centres, (ii) the distance between the Arg Cbeta and Asp Cbeta atoms, and (iii) the pseudo-dihedral angle defining the Arg and Asp side-chain orientation formed by the Arg Czeta, Arg Calpha, Asp Calpha and Asp Cgamma atoms. A comparative conformation-activity study was performed between linear RGD peptides and strongly constrained cyclic (S,S) -CDC- bearing compounds, which cover a wide range of inhibition potency of platelet aggregation. It is concluded that the fulfilment of the -45 degrees < or = pseudo-dihedral angle < or = +45 degrees criterion is a prerequisite for an RGD compound to exhibit inhibitory activity. Once this criterion is accomplished, the longer the distance between the charged centres and/or between the Arg and Asp Cbeta atoms, the higher is the biological activity. In addition, the stronger the ionic interaction between Arg and Asp charged side chains, the lower the anti-aggregatory activity.

[Show abstract][Hide abstract]ABSTRACT: The Arg-Gly-Asp RGD motif of adhesive proteins is recognized by the activated platelet integrin alpha(IIb)beta3. Binding of fibrinogen (Fg) to activated alpha(IIb)beta3 causes platelet aggregation and thrombus formation. Highly constraint cyclic (S,S) -CXaaC- containing peptides incorporating the (S,S) -CDC- and (S,S) -CRC- motifs were tested for their ability to inhibit platelet aggregation and Fg binding. Our results suggest that the above cyclic scaffolds stabilize a favorable structure for the antiaggregatory activity (IC50-values ranged from 1.7 to 570 microm). The peptides inhibited Fg binding with IC50-values up to 30-fold lower than those determined for the inhibition of the adenosine diphosphate (ADP)-induced platelet aggregation. Importantly, peptides (S,S) PSRCDCR-NH2 (peptide 11) and (S,S) PRCDCK-NH2 (peptide 10) did not inhibit PAC-1 binding to the activated platelets at a concentration in which they completely inhibited Fg binding. Moreover, (S,S) PSRCDCR-NH(2) (peptide 11), one of the more active peptides, inhibited ADP-induced P-selectin exposure. By contrast, peptide (S,S) Ac-RWDCRC-NH2, incorporating the inverse (S,S) -DCRC- sequence (peptide 16), failed to inhibit P-selectin exposure whereas at the same concentration, it effectively inhibited PAC-1 and Fg binding. It is concluded that peptides containing the (S,S) -CDC- as well the (S,S) -CRC- sequences, exhibit a broad range of activities toward platelets, and could be helpful tools for elucidating the structural interaction of Fg with the integrin receptor alpha(IIb)beta3, in its activated form. Furthermore, the (S,S) -RCDC- sequence can be used as a scaffold for developing potent non-RGD-like Fg-binding inhibitors.

[Show abstract][Hide abstract]ABSTRACT: The platelet integrin receptor alphaIIbbeta3 plays a critical role in thrombosis and haemostasis by mediating interactions between platelets and several ligands, primarily fibrinogen. We have previously shown that the synthetic peptide YMESRADRKLAEVGRVYLFL corresponding to residues 313-332 of alphaIIb, is a potent inhibitor of platelet aggregation and fibrinogen binding to alphaIIbbeta3, interacting with fibrinogen rather than the receptor. Furthermore, we have demonstrated that the biological activities of the above peptide are due to the sequence YMESRADR, which corresponds to residues 313-320. By using new synthetic peptide analogues we investigated the structural characteristics responsible for the biological activity of YMESRADR as well the possible influence of the adjacent amino acids on the peptide's biological potency. According to our results, the synthetic octapeptide YMESRADR, is a potent inhibitor of platelet aggregation and P-selectin expression. Furthermore, YMESRADR inhibits fibrinogen binding but it does not significantly influence the binding of PAC-1 to ADP-activated platelets. The inhibitory potency of YMESRADR was gradually diminished by deleting the YMES sequence from the amino terminus and prolonging the carboxyl terminus of this peptide with the KLAE sequence. Extension of YMESRADR towards the amino terminus with the GAPL sequence (GAPLYMESRADR) does not modify the biological activity of YMESRADR. Furthermore, extension of GAPLYMESRADR at its carboxy terminus with the KLAE sequence (GAPLYMESRADRKLAE) significantly diminished its biological potency. Substitution of E315 with D significantly enhances antiaggregatory potency and completely abolishes the inhibitory effect on P-selectin expression. Importantly, the D315-containing peptides inhibit to a similar extent both fibrinogen and PAC-1 binding to activated alphaIIbbeta3 in contrast to the E315-containing peptide which only inhibits fibrinogen binding. In conclusion, the present study suggests that the YMESRADR sequence 313-320 of alphaIIb, is an important functional region of the insert connecting the beta2 and beta3 antiparallel beta-strands of the W5 blade of the alphaIIb subunit. Structural changes significantly modify the biological properties of this region.

Note: This list is based on the publications in our database and might not be exhaustive.

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INTRODUCTIONInformation on the favored conformational states ofshort linear peptides comes mainly from x-ray diffrac-tion, NMR studies in different solvents, and molecularmodeling calculations.1–3For instance, five differentcrystal structures for Leu-enkephalin and three forMet-enkephalin, depending on solvent crystallizationconditions, have been reported.4,5By taking advan-tage of the solubility of peptides in a number of polarsolvents, various solvent-dependent conformationalstates can be resolved. It is generally accepted thatthese states are found in very fast conformationalexchange. The structures of peptides, especially thosederived from NMR, are believed to represent onlypopulation-weighted averages over all conformers ina given solvent at ambient temperature. Only slowtransitions in the NMR time scale conformational,such as the amide bond cis–trans interconversion,have been resolved to date for linear peptides byNMR spectroscopy. To study very rapid processes,such as the folding of peptides and proteins, newmethods had to be developed (stopped-flow fluores-cence, stopped-flow CD, temperature jump, etc.),which allow the earliest events in folding to beprobed.6,7Using temperature jump and photodissocia-tion techniques, Eaton et al.8have shown that some?-helices are formed in a few nanoseconds, whereasothers require microseconds to fold, depending on theparticular amino acid sequence. To date, intermediateconformational states between folded and unfoldedstates in peptides have not been detected.Some recent studies have indicated that short linearpeptides can adopt different conformational states indimethylsulfoxide (DMSO) solutions depending onthe pH value of the aqueous solution they originatedfrom.9–12We have also provided experimental evi-dence by17O-NMR spectroscopy for slow conforma-tional exchange of Boc–[17O]Tyr(2,6-diClBzl)–OH(Boc: tert-butoxycarbonyl; Bzl: benzyl) in DMSOsolution.13Our conclusion was based on the detectionof two, rather than one,17O resonances for the car-boxyl group of the peptide in DMSO, most probablydue to the engagement of the carboxyl group in astrong hydrogen-bonding interaction. In CDCl3solu-tion, on the other hand, a single17O resonance, re-sulting from the fast exchange between open andhydrogen bonded states, was observed. The depen-dence of the peptide conformation on the nature of thereconstitution medium has also been highlighted bythe recent work of Boden et al., who studied thethree-dimensional (3D) structure of a linear 27-resi-due peptide in lipid bilayers by Fourier transforminfrared (FTIR) absorption.14When that peptide wasreconstituted from methanol, it adopted a ?-strandstructure, while in the case of 2,2,2-trifluoroethanol itformed initially an ?-helix, which relaxed very slowly(within hours) to an equilibrium state between ?-helixand ?-sheet.It appears, therefore, that the nature of the solventand the conditions employed in the conformationalreconstitution might influence the prevalence of acertain peptide conformation.The work reported here aims to develop a NMR-based strategy that would allow us to identify directlydiscrete conformational states of short linear peptides,differentiate them from the average one, and gain newinsight into their folding process. The tripeptide Ac–RGD–NH2(Ac–Arg–Gly–Asp–NH2, Ac: acetyl) wasused as a model compound for our NMR and molec-ular modeling studies.15–18Solutions of this peptide inDMSO-d6, reconstituted from aqueous solutions atdifferent pH values, were studied by1H-NMR spec-troscopy at several temperatures (in the range 300–355 K). We managed to detect discrete conforma-tional states of the peptide in DMSO-d6, which varywith the initial pH of the solution, and to show thatthese states can be in slow exchange depending on thereconstitution conditions. We present here our find-ings and discuss their implications for peptide folding.MATERIALS AND METHODSReagents and solvents were used without further purifica-tion.2-(1H-benzotriazol-l-yl)-1,1,3,3-tetramethyluronium(TBTU), 1-hydroxybenzotriazole (HOBt), and Boc aminoacids were purchased from Neosystem (France); solventsfrom Labscan Ltd. (Ireland); trifluoroacetic acid (TFA) anddiisopropylethylamine (DIEA) from Merk Schuchardt (Ger-many); and 4-methyl-benzhydrylamine resin (MBHA) resinfrom Saxon Biochemicals (Hannover, Germany). DMSO-d6and tetramethylsilane (TMS) were purchased from Euriso-top (France).Synthesis of Ac–RGD–NH2This was carried out by the stepwise solid-phase procedureon a MBHA resin following the Boc chemistry.19Arg wasintroduced as Boc–Arg(Tos)–OH (Tos: toluene-4-sulfonyl)and Asp as Boc–Asp(OBzl)–OH. Coupling reactions wereperformed using the molar ratio of amino acid/TBTU/HOBt/DIEA/resin 3/2.9/3/3/1. The ?NH2 group, aftercleavage of the Boc protecting group with TFA, was acety-lated using an excess of Ac2O in pyridine (the ratioAc2O/—NH2group was 30:1). Ac–RGD–NH2was cleavedfrom the resin with anhydrous HF in the presence of phenoland anisole as scavengers. The crude material (yield 80%)was subjected to high performance liquid chromatographyThe Ac–RGD–NH2Peptide73

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(HPLC) purification (semipreparative reverse-phase C18col-umn) using gradient elution with the following solvents: A,H2O/0.1% TFA; B, CH3CN/H2O/TFA (10/90/0.1). A pro-grammed gradient elution (4 mL/min) was applied (A/B: 90/10–A/B: 75/25), elution time 20 min (yield 60%). The purityof the peptide was checked by analytical HPLC and the correctmolecularmasswasconfirmedbyelectrosprayionizationmassspectroscopy (ESI-MS) (MW calc.: 387.40; found: 387.62).Synthesis of Ac–RGd–NH2This was carried out on a MBHA resin according to Bocchemistry as described above. The purity of the peptide waschecked by analytical HPLC and the correct molecular masswas confirmed by ESI-MS.1H-NMR ExperimentsThe NMR samples were prepared by dissolving the solidmaterial in H2O, and adjusting the pH to the desired valuewith NaOH or HCl. The aqueous solutions were lyophi-lized, and weighted amounts were dissolved in DMSO-d6atconcentrations ?5 mM. The NMR experiments were per-formed at 295–355 K on Bruker AMX 400 and Avance 500spectrometers.Thestandard(COSY), total COSY (TOCSY), and rotating frame nuclearOverhauser effect spectroscopy (ROESY) Bruker micropro-grams were used. The TOCSY spectra were recorded usinga mixing time of 100 ms. The spectral width in F1 was 5600Hz. Various ROESY experiments were performed usingmixing times of 250 and 350 ms at 300, 305, and 310 K.The rate constants of rotation about the guanidinium N?–C?(k?), C?–N?1(k?1), and C?–N?2(k?2) bonds were obtained bycalculating the NMR line shape for a four-site exchange pro-cess using the general multiple site exchange matrix algo-rithm.20The best fitted simulated spectra were obtained byusing the spectral parameters (chemical shifts, line widths, andintensities) for guanidinium protons and varying rate constantsk?, k?1, and k?2. The natural line widths of guanidinium NHsignals required in these determinations were estimated bymeasuring the line widths of nonexchanging Arg amide protonsignals at appropriate temperatures. All calculations were per-formed using the program Muses (MUltiple Site ExchangeSimulations).21The activation parameters were evaluated fromEyring equations:correlationspectroscopyln?k/T? ? 23.76 ? ??H*/RT? ? ??S*/R?and?G* ? RT?23.76 ? ln(k/T?]where R is the universal gas constant.Structure CalculationStructure calculation was carried out using the software DY-ANA (DYnamics Algorithm NMR Applications).22The dis-tance restraints used as inputs in DYANA were derived fromareconstituted in DMSO-d6after lyophilization from an aque-ous solution at pH 4.9. The ROESY spectrum was recorded at310Kwithamixingtimeof250ms.TheROEintensitieswereconverted into distances using the ?1-?2 cross peak of Asp asreference. Thirty-six upper-limit cross-peak intensities, classi-fied as strong (up to 2.8 Å), medium (up to 3.5 Å), and weak(up to 5 Å), were used as input restraints for the calculation.Appropriate corrections for center averaging were added toDYANA restraints for degenerate proton resonances.23Nolower limits were used. Constraints for ?, ?, and ?1angleswere calculated using the HABAS program of DYANA pack-age. Six3J??2and3J?coupling constants were included witha tolerance of 2.5 Hz. All distance and angle constraints wereassigned the default relative weight of 1. The default toleranceof 0.05 at target function units was applied. The calculationswere performed using the standard minimization protocol andthe REDAC (REdundant Dihedral Angle Constraints) strategyimplemented in DYANA.1H-1H ROESY spectrum of the Ac–RGD–NH2peptideRESULTS AND DISCUSSIONConformational State of Ac–Arg–Gly–Asp–NH2, Reconstituted in DMSO-d6from an Aqueous Solution at pH 2.0The complete assignment of all proton resonances ofAc–Arg–Gly–Asp–NH2was based on the combineduse of COSY, TOCSY, and ROESY experiments. The1H-NMR spectrum in DMSO-d6solution of the pep-tide reconstituted after lyophilization from an aqueoussolution at pH 2 is shown in Figure 1 (bottom). Theresonance at 12.3 ppm (not shown) confirms the pro-tonated state of the Asp ?-carboxylic group. The highabsolute temperature coefficient values of all of theNH protons, including the C-terminal amide protons(?8 ppb/K), suggest that they are exposed to thesolvent. The equal3JN?and3JN??values (5.32 Hz) ofGly indicate that there is a free rotation about theNOC?bond of this residue,24while the Asp3J??and3J???coupling constant values (5.27 and 8.46 Hz)correspond to a high percentage (?80%, Table I) ofthe two energetically favored C?–C? rotamers25–28(Iand II, ??rotational restrictions about the C?OC?bond. On theother hand, the Arg–N?H and Arg–N2symbology indicates both N?atoms and all four pro-tons linked to them) proton resonances, at 7.63 and7.13 ppm, respectively, are attributed to their nonhy-drogen-bonded states.10,11,29It must be noted that theArg–N2can be detected as two broad peaks due to chemicalexchange in the guanidinium group.10,11,30,31In thisconformational state two separate, broad peaks at 7.291?60°, 180°), suggesting the absence of?H4(this latter?H4protons under free rotational conditions74Biris et al.

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and 6.91 ppm, respectively, were detected at 295 K(data not shown), which collapse to a broad peak at300 K (Figure 1) at 7.13 ppm. Strong sharpening ofthis peak is observed as the temperature increases to355 K (data not shown). The NMR data thus suggestthat Ac–Arg–Gly–Asp–NH2is found in the extendedconformational state when lyophilized from aqueoussolution at pH 2 and redissolved in DMSO-d6.NMR spectra recorded back at 300 K after heatingthe sample to 355 K and keeping it at 300 K for varyingtime periods reveal the presence of a second set ofresonances indicative of a slow aggregation process tak-ing place under these conditions (data not shown). Thisconclusion is supported by the appearance in the ES-MSspectrum of a low intensity peak originating from asmall amount of the dimeric form of the peptide. Inter-estingly, this peak was not detected under neutral orbasic conditions (data not shown). Sanderson et al.32reported a similar observation for the (SS) Mba–Arg–Gly–Asp–Man peptide (Mba: 2-mercaptobenzoate;Man: 2-mercaptoanilide) in DMSO-d6solution. Theseauthors, based on the fact that the Arg–N?H was notdetected in the second set of resonances, concluded thatsuch a behavior could be the result of a slow hydrolyticprocess. This phenomenon can be excluded in our caseby the full set of resonances for the Ac–Arg–Gly–Asp–NH2peptide present in the one-dimensional (1D) NMRspectrum and in the TOCSY and ROESY NMR spectraas well.Conformational State of Ac–Arg–Gly–Asp–NH2, in DMSO-d6Reconstitutedfrom an Aqueous Solution at pH 3.3The ionization state of the peptide remains the sameas that at pH 2.0, as indicated by the presence of abroad peak at 12.25 ppm corresponding to the pro-FIGURE 1Ac–RGD–NH2in DMSO-d6solution at various temperatures. Conformational reconstitution of thepeptide was performed from an aqueous solution at pH 3.3. For comparison the spectrum of thepeptide reconstituted from an aqueous solution at pH 2.0 is given (bottom). Arg–N2indicates four protons.NH (A), C?H (B), and C?H (C) regions of the 400 MHz1H-NMR spectra of?H4notationThe Ac–RGD–NH2Peptide75

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tonated form of the Asp ?-COOH group. Neverthe-less, the dramatic changes in the resonance frequen-cies and the line shapes over the entire spectrum areindicative of a new conformational state of the pep-tide. Thus, the Asp–NH proton broadens and is down-field shifted from 8.19 to 8.46 ppm, the Arg–N?Hbecomes very broad and vanishes into the baseline at300 K (Figure 1A), the Arg–N2a broad peak at 7.10 ppm, and the two carboxamideprotons are seen as a sharp resonance at 7.10 ppm anda broad one at 6.92 ppm. The line shape of theresonances can provide information about the inter-acting parts of the molecule in the new conforma-tional state. The observed broadening of many reso-nances is not due to a change in the rotational corre-lation time of the entire peptide since not all of theresonances are broadened to the same extent and thenarrowing of each resonance occurs at different tem-perature values (Figure 1). Thus, the difference in theline width broadening of the Gly–NH, Asp–NH, Asp–C?H resonances, the upfield shifted Gly–C?H, Asp–C?H, and the C-terminal amide proton resonances, aswell as the downfield shifted Arg–C?H, indicate theoccurrence of a slower exchange process, which af-fects mainly these groups, compared to the other partsof the molecule. It is also interesting to note that fromthe geminal Gly–C?H2and Asp–C?H2protons, onlythe resonances of the upfield shifted protons appearbroadened with that of the Asp–C?H proton almostvanishing into the baseline (Figure 1C). Since theAsp–C?H, Gly–C?H, Asp–C?H, and the Arg–C?Hprotons cannot be involved in any other exchangenH4protons appear asprocess, we assume that the origin of their broadeningresides in a conformational exchange process. It isalso clear that the Gly–C?H2and Asp–C?H2groupsare not involved directly in the exchange process.Therefore, the differential broadening of their upfieldshifted protons must originate from conformationalinterconversion of adjacent groups, which affectstheir local magnetic environment. Bogusky et al.33have reported a similar broadening for the Gly–C?H2protons brought about by freezing to ?80°C a meth-anolic solution of a cyclic (S,S) CRGDC peptide witha viscosity similar to that of DMSO at room temper-ature. In this case, molecular dynamics simulationshave shown the presence of conformers differing byrotational inversions of the peptide-bond planes be-tween the Arg–Gly and Gly–Asp residues.The very fast interconverting rate between the con-formers builds up gradually by raising the temperaturefrom 300 to 350 K (Figure 1). The first groups of themolecule to achieve the very fast interconverting rateare located around Gly (at 310 K, Figure 1B). Mostnotable is the fact that at 330 K the upfield shiftedAsp–C?H and especially the Arg–N?H protons arestill undergoing considerably slower exchange as re-vealed by their resonance broadening (Figures 1A,1C, and 2). This finding probably indicates that thereis a contact between the Arg–N?H proton and the Asp?-COOH group responsible for this conformationalexchange.Besides the chemical shift changes observed be-tween the conformational states reconstituted from pH2.0 and 3.3, respectively, the temperature coefficientTable IProtons of Ac–Arg–Gly–Asp–NH2in DMSO-d6Solution at 300 K, Reconstituted from Aqueous Solutionsat Various pH ValuesChemical Shift Differences of the Geminal Gly–C?H2, Asp–C?H2, Arg–C?H2, Arg–C?H2, and Arg–C?H2ConformationalReconstitutionSolvent?? (ppm)Arg–C?H2Arg–C?H2Arg–C?H2Gly–C?H2Asp–C?H2Rotamersaof theAsp C?–C?Bond (%)IIIIIIH2O, pH ? 2.0H2O, pH ? 3.3H2O, pH ? 4.0H2O, pH ? 4.4H2O, pH ? 4.90.150.250.360.410.47?0.000.030.040.080.08?0.000.060.110.160.170.080.080.200.280.300.160.16b0.35c0.450.5126251454312225242044b64c70d7155aRotamer I (?1? ?60°), II (?1? 180°), III (?1? 60°). The values of Jgand Jtused for estimation of the rotamer populations were 2.32and 13.70 Hz, respectively.bMeasured at 340 K.cMeasured at 330 K.dMeasured at 310 K.76Biris et al.